Summary Electron microscopy (EM) has become firmly established in the armamentarium of tools available to structural biologists, providing three-dimensional reconstructions of components of the cell across a wide range of size scales. Our vision for this technique is to provide a straightforward manner in which users can proceed from raw data to reliable 3D reconstructions through a pipeline that both facilitates management of the processing steps and makes the results at each step more transparent. To this end we have developed Appion, a modular and transparent pipeline that integrates and provides interoperability between disparate software applications and procedures. The user manages and controls the software modules via a web-based interface, and all results are similarly available using web-based viewers directly linked to an underlying database that tracks all processes and data and provides communication between the software modules. Initial dissemination to the EM structural biology community was enabled by support provided by ARRA funding and here we request funds to provide continued support for the development and maintenance of Appion and to make it accessible to a much larger community of users. An essential aspect of this project is to ensure that Appion can be readily and continuously extended by the community to accommodate new software packages and algorithms as they become available. We have strong support from the EM community for the development of Appion and, very importantly, most of the major EM software developers support having their packages integrated into Appion, and distributed with the Appion installation. This community support and cooperation provides a unique opportunity to establish long needed software standards and interoperability for our community. Appion ensures that the most commonly used EM tools are readily accessible and also provides a way for developers of new tools to integrate them into an existing pipeline. This will bring much needed transparency to what is currently available and how to build on existing tools using a standardized vocabulary and data flow. Our specific aims are centered on (i) improving Appion portability across operating systems, and high-end computing environments; (ii) improving extensibility by simplifying methods for adding new modules to the software; (iii) ensuring the continued evolution of Appion by improving methods for updating and optimizing the underlying databases; (iv) improving the usability by extending and simplifying the installation process and improving the graphical user interface; (v) providing more extensive and comprehensive testing essential to ensuring the accuracy of the scientific results produced by the pipeline. All these efforts will of course be accompanied by the development of comprehensive documentation and tutorials. PUBLIC HEALTH RELEVANCE: Electron microscopy (EM) is now established as an essential tool for studying macromolecular machines that are central to cellular function, and thus has a basic and fundamental relevance for both the healthy and diseased states. EM methods require access to an array of sophisticated computational tools and procedures, which presents a barrier to entry for newcomers and a potential source of errors. This project will provide unified access to an integrated set of interoperable tools that are easy to use, easy to adapt and extend, and fully documented.